Characterization Of An Autoimmune Disease-associated Functional Variant Rs6927172 In Regulating Gene Expression Of TNFAIP3 And IL-20RA

Background:Autoimmunity cause damage of macromolecular substances,tissue or cell,which inturn results in organ,system or even systemic dysfunction,causing clinical manifestations,that is autoimmune disease(autoimmune disease,AID).Presently,the vast majority of autoimmune diseases cannot be cured.With aging,the function of affected organ or system gradually declines until the function is lost,eventually leading to the death of the patient.Human genetic association studies have identified many single nucleotide polymorphisms(SNP)consistently associated with various autoimmune diseases.Interestingly,most of autoimmune disease-associated SNP(90%)map to non-coding regions of the human genome.The chromosomal region 6q23 is an important and incredibly complex non-coding genomic locus in autoimmunity.The locus contained various regulatory elements and implicated in multiple diseases.A tag SNP rs6920220 at the chromosomal region6q23 has been associated with rheumatoid arthritis(RA),systemic lupus erythematosus(SLE),celiac disease(CeD),type 1 diabetes(T1D),and inflammatory bowel disease(IBD).The SNP rs6920220 is in strong linkage disequilibrium(LD)(r~2>0.8)with eight other SNP(rs6933404,rs62432712,rs2327832,rs928722,rs35926684,rs6927172,rs11757201 and rs17264332)(Chr:6 137,638,098-137,685,367).However,the true causal variant at this locus and the underlying molecular mechanism by which the causal variant influences autoimmunity remains elusive.The association signals have been explained to the TNFAIP3 gene,the most likely causal gene within the locus.TNFAIP3 has potent anti-inflammatory function and played a key role in restoring cellular homeostasis through NF-?B inhibition as related to the ubiquitin editing and ubiquitin binding activities,the A20 protein is now recognized as a central inhibitor of inflammatory responses.Accumulating evidence suggested that multiple non-coding variants at the TNFAIP3 gene locus in transcriptional regulation through long-range chromosomal interactions.In addition to TNFAIP3,there are interferon gamma receptor 1(IFNGR1),oligodendrocyte transcription factor 3(OLIG3)and interleukin 20 receptor A(IL-20RA)around the these 8 SNPs.whether these genes are also involved in the regulation of autoimmunity with TNFAIP3,is also a scientific problem to be solved.Method:We used bioinformatics methods to analyze the potential biological functions of 8 SNPs and knocked out rs6927172 element using clustered regularly interspaced short palindromic repeats/associated protein 9(CRISPR/Cas9)technology.With improved Transcription activator-like effector nucleases(TALEN)technology,we achieved to specifically bind to rs6927172 elements.We also exploring the interaction between rs6927172 element and the target gene using Chromatin conformation capture followed by qPCR,3C-qPCR).Using gel electrophoresis or electrophoretic mobility shift assay(EMSA)techniques to assess the binding of rs6927172 elements to intracellular transcription factors.Finally,Western Blot and qPCR were used to detect the gene expression.Results:The rs6927172 locus was modified by histone methylation and had binding activity to a large number of transcription factors.To further clarify the potential biological function of rs6927172,we generated a knockout of the intergenic SNP rs6927172 using CRISPR/Cas9 system in embryonic kidney cells transformed with large T antigen(HEK293T).The genetic variant rs6927172 lies upstream of the TNFAIP3 gene.Therefore,we assessed expression of genes in the genomic locus:chr6:137,300,000-138,300,000.This region includes four genes(IL-20RA,IL-22RA2,IFNGR1,OLIG3)upstream of the variant and the TNFAIP3 gene.Furthermore,identification of transcription factors binding to the SNP rs6927172 is another critical step to better understanding the role of the variant in regulating expression of its targeted genes.In this study,we develop a new use of the TALE protein(TALENs without the nuclease domain)to dissect EMSA and further assess the role of the variant rs6927172 in influencing the expression of its targeted genes.The results showed that knocking out the rs6927172 locus or blocking the binding of rs6927172to transcription factors affected mRNA and protein expression of multiple genes,including IL-20RA and TNFAIP3.We also used 3C-qPCR technique to detect the frequency of interaction between the sequence of rs6927172 and the target gene.The results showed that knocking out the rs6927172 locus or blocking the binding of rs6927172 to transcription factors can reduce the frequency of interaction between rs6927172 and the promoter of IL-20RA and TNFAIP3 gene.Conclusion:we applied CRISPR/Cas9-mediated knockout and the new use of the TALE targeting variant rs6927172 to clarify the role of rs6927172 in autoimmune disease gene TNFAIP3,and discovered IL-20RA as an potential pathogenic gene in autoimmune disease.provide new evidence for the variant as a causative variant for multiple autoimmune diseases.Previous studies have suggested that the eight SNPs in this group with strong linkage disequilibrium affect the susceptibility of autoimmune diseases by regulating the function or expression of the TNFAIP3 gene.In this study,rs6927172 was identified as a functional SNP locus by knockout experiments,and it was also proved that this group of SNPs not only affected the expression of TNFAIP3gene,but also affected IL-20RA gene expression,suggesting IL-20RA as an autoimmune disease susceptibility gene.It also shows that the SNP site is a long-range regulator that regulates the expression of multiple target genes.TALEN is a genome editing tool.We have for the first time modified the TALEN expression system and successfully expressed a TALE protein that has only DNA binding function and no DNA cleavage activity at the cellular level.Expression of TALE protein in cells can bind to rs6927172 elements and block the binding of other transcription factors,further demonstrating that target genes of this SNP functional domain include TNFAIP3 and IL-20RA.Finally,this study used the 3C-qPCR to further elaborate the molecular mechanism of the functional SNP regulating two target genes.The intensity of its interaction with the promoters of the two target genes was quantified,and it was further confirmed that knocking out the SNP and overexpressing the TALE can directly affect the interaction frequency of the SNP and the target gene promoter.